Public consultation. Question time.
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Communication↗
Biomedical subjects
P Edwards
Publications and source records attributed to P Edwards.
Crystal structure of beta-ketoacyl-acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes.
In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution. The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices. The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha. The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis. The two active sites are located near the dimer interface but are approximately 25 A apart. The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues. In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Using radiology records to improve epidemiological information in paediatric fractures: a feasibility study.
OBJECTIVES: To assess the feasibility of using routine computerised radiology records for community injury surveillance data using fractures in the child population as an example. DESIGN: Radiology and in-patient computerised files were accessed to extract information concerning type of fracture, age, sex, and home address. Diagnostic coding of radiological report was carried out using the ICD-9 classification. Children were assigned to local authority wards using home postcodes derived from home addresses. Ward fracture rates were calculated using 1991 census data. The association between ward fracture rates and deprivation was explored using Townsend scores. SETTING: North Tyneside General Hospital. SUBJECTS: Children age 10-14 y receiving care as in-patients or out-patients for long-bone fractures. RESULTS: Between April 1991 and March 1996 a total of 497 long-bone fractures were identified. Fractures in boys exceeded those in girls by a ratio of 2:1. The most common fracture identified was of the radius and ulna. There was no evidence of an ecological association between long-bone fracture rates in children aged 10-14 y and social deprivation. CONCLUSIONS: Computerised radiological records may be used to improve epidemiological information concerning fractures. However, at present, considerable time and effort is required to access the information, to identify and to classify, long-bone fractures. Such data could be used to assist in the audit of clinical care and long-term outcomes, and to inform effective local planning and evaluation of injury prevention initiatives.
Adolescent↗
KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme.
cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18.1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Restored gap junctional communication in non-tumorigenic HeLa-normal human fibroblast hybrids.
Gap junctional intercellular communication (GJIC) has been implicated in homeostasis, development, differentiation, wound healing or regeneration and adaptive responses of differentiated cells. The dysfunction of homologous or heterologous GJIC has been associated with the tumorigenic phenotype. Restoration of growth control and the suppression of the tumorigenic phenotype have been previously associated with the up-regulation of GJIC by various anti-tumorigenic chemicals or transfection of connexin genes into tumor cells. To test the hypothesis that 'tumor suppressor' genes may be associated with the up-regulation of GJIC, we tested clones of tumorigenic HeLa, several non-tumorigenic HeLa-normal human fibroblast somatic cell hybrids and a tumorigenic segregant of one of the non-tumorigenic hybrids for GJIC. The parental HeLa cells (D98 AH.2) had no detectable GJIC but expressed detectable connexin 43 transcripts, while the non-tumorigenic HeLa-human fibroblast hybrids, which contained the chromosome 11 from the normal human fibroblast (CGL-1, CGL-2, ESH15 and EHS15c1), expressed ample connexin 43 transcripts and showed proficient GJIC. The tumorigenic segregant (CGL-3) from the non-tumorigenic HeLa-human fibroblast hybrid showed no GJIC or connexin 43. These results show that the presence of GJIC is closely linked to the suppression of the tumorigenic phenotype in the HeLa-human fibroblast hybrid and further suggest that GJIC may be associated with the mechanisms of tumor suppression. The mechanism by which the tumor suppressor gene(s) on the normal chromosome in the HeLa-human fibroblasts induces the up-regulation of connexin 43 is not yet explained.
Base Sequence↗
On the meaning of "sensitivity": a rejoinder.
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Clinical Chemistry Tests↗
Using the Internet to support self-care.
Nurses have long recognized the importance of self-care. In the current Information Age, nurses can use the Internet to locate computer- and non-computer-based programs and tools that encourage people to become active consumers who control decisions and actions related to their health. The Internet can support self-care in two main ways: by supplying information and by providing a medium for interactive social support. But there are pros and cons to such use of the Internet.
Humans↗
Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli.
Analysis of the beta-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene. Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene. This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF. We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
On the meaning of "sensitivity".
The term "sensitivity" (as applied to an analytical method's performance) has again become a subject of controversy. Certain authorities (e.g., IUPAC) define a system's sensitivity as the response curve slope (or response/dose), others (e.g., IFCC) in terms of the detection limit. Many investigators have failed to perceive the contradiction between these concepts, wrongly assuming that maximizing "sensitivity" in the first sense maximizes it in the second (i.e., that they are inversely related). The existence of different meanings for this term (when used in the present context) is a source of confusion that has, among other things, led to erroneous ideas relating to immunoassay design. Such confusion should be terminated by adoption of one or the other of the definitions. However, the definitions are not of equal merit. We advance arguments against retention of the "slope" definition, which conflicts with the word's common meaning and is meaningless as an indicator of the performance of a measuring system.
Clinical Chemistry Tests↗
Managing HIV. Part 5: Treating secondary outcomes. 5.3 HIV, gastrointestinal and hepatobiliary disease.
Diseases of the gastrointestinal tract, biliary tree and liver are a major source of morbidity and mortality in patients with HIV infection, who may present with a complex array of signs and symptoms that require accurate diagnosis and therapy.
Biliary Tract Diseases↗
Tumor cell nitric oxide inhibits cell growth in vitro, but stimulates tumorigenesis and experimental lung metastasis in vivo.
Arginine-derived nitric oxide (NO) has been identified in some tumor cell lines and solid human tumors. The effect of tumor cell NO on tumor biology is poorly understood. The purpose of this study was to investigate the effect of NO production by EMT-6 murine breast cancer cells on tumor cell growth in vitro and subcutaneous tumor growth and experimental pulmonary metastasis in vivo. EMT-6 cells were incubated with endotoxin (LPS, 10 microgram/ml) and interferon-gamma (IFN, 50 U/ml), in the presence or absence of the NO synthase inhibitor, omega-nitro-L-arginine methyl ester (L-NAME, 2 mM), and NO production and cell number were assessed 24 hr later. EMT-6 cells were also treated overnight with LPS/IFN, in the presence or absence of L-NAME, washed and injected either subcutaneously in the dorsal flank (n = 40) or via the tail vein (n = 40) of syngeneic BALB/c mice. Two weeks following tumor cell injection, tumor size and number of pulmonary metastases were assessed. LPS/IFN stimulated NO production in EMT-6 cells and inhibited cell growth in vitro by 50%. L-NAME blocked LPS/IFN stimulation of NO production and restored cell growth to near control levels. When injected into BALB/c mice, LPS/IFN-stimulated tumor cells demonstrated a two-fold increase in subcutaneous tumor growth and experimental pulmonary metastases over control cells. L-NAME reduced tumor size and number of lung metastases to control levels, suggesting that tumor cell NO production was responsible for this effect. In summary, LPS/IFN-stimulated NO production in EMT-6 tumor cells inhibits tumor cell growth in vitro, yet paradoxically augments tumor growth and metastasis in vivo.
Analysis of Variance↗
Enhancement in antigen binding by a combination of synergy and antibody capture.
The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen, binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.
Animals↗
An unusual case of testicular pain.
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Cysts↗
Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.
The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.
Amino Acid Sequence↗
The abdominal compartment syndrome: the physiological and clinical consequences of elevated intra-abdominal pressure.
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Abdomen↗
Massive choroidal hemorrhage in age-related macular degeneration: a complication of anticoagulant therapy.
BACKGROUND: Age-related macular degeneration (ARMD) is the leading cause of legal blindness in the elderly, causing profound loss of central vision. METHODS: A 90-year-old patient with exudative age-related macular degeneration who had been placed on anticoagulant therapy for pulmonary emboli experienced a massive choroidal hemorrhage and retinal detachment. Angle closure glaucoma was precipitated by forward movement of the iris-lens diaphragm and vision was lost in the eye. The patient was subsequently removed from anticoagulant therapy, although he had potentially fatal bilateral pulmonary emboli, because of the patient's concern for the quality of his remaining life. RESULTS: This case report is intended to alert optometrists to the ocular complications of anticoagulant therapy, particularly the risk of massive choroidal hemorrhage in anticoagulated patients with exudative ARMD. CONCLUSIONS: An optometrist's responsibility to inform primary care providers of the potential ocular complications of anticoagulant therapy for patients with exudative ARMD is also described.
Aged↗
Intraoperative graft tensioning alters viscoelastic but not failure behaviours of rabbit medial collateral ligament autografts.
The effects of three different degrees of intraoperative graft tensioning on measures of ex vivo laxity, viscoelastic behaviour, and structural and material failure of isolated healing medial collateral ligament autografts were investigated in a rabbit model. The grafts were orthotopically replaced at one of three different loads (too tight, anatomic, or too loose) and were mechanically evaluated after 0, 12, 24, and 48 weeks of healing. Laxity of the ligament was influenced by intraoperative graft tensioning at time zero. However, after 12 weeks of healing, values for laxity were indistinguishable among the experimental groups. Cyclic load relaxation, a measure of viscoelastic behaviour, was significantly influenced by intraoperative graft tensioning, and this effect persisted even after 48 weeks of healing. Grafts placed under excessive tension relaxed one-third less than grafts placed under abnormally low in situ tension. The relevance of these differences remains to be determined. Intraoperative tensioning had no significant influence on characteristics of structural or material failure of the graft during the first year of healing. These results suggest that, in this model, control of graft tension at the time of placement and fixation does not improve the failure characteristics of the medial collateral ligament. The structural strength of the grafts collectively improved to nearly normal values after 48 weeks; however, material recovery was less complete. Failure loads averaged 89% of control values, whereas failure stress averaged only 52% after 48 weeks of healing.
Analysis of Variance↗
Monitoring pathological assembly of tau and beta-amyloid proteins in Alzheimer's disease.
This double-labelling confocal microscopy study of the neuropathology of Alzheimer's disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects beta-amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse beta-amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tangle-bearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for beta-pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.
Aged↗